A fundamental property of life is the ability to replicate itself. Researchers have now created the first molecules of RNA, DNA’s singled-stranded relative, that are capable of copying almost any other RNAs. The discovery bolsters the widely held view among researchers who study the origin of life that RNA likely preceded DNA as the central genetic storehouse of information in the earliest cells some 4 billion years ago. Ironically, the new RNA copiers still can’t duplicate themselves. But if future souped-up versions can pull that off, it could do more than reinforce notions of RNA’s primordial role—it could lead to the creation of the synthetic modern microbes that use RNA as their sole source of genetic information.
In order to grow and replicate, all modern cells require DNA, RNA, and proteins, and the synthesis of each inside cells requires the other two. Researchers in the 1960s hypothesized that modern cells evolved from progenitors that didn’t require this interdependence. RNA seemed a likely first biomolecule, because, like DNA, it can store information, and, like proteins, it can act as a catalyst to speed up certain chemical reactions. Researchers also discovered early on that RNA is at the core of several modern enzymes critical to life, such as the ribosome that builds proteins. So some scientists hypothesized life that started as an “RNA world”—a period in which RNA controlled both the genetics and biochemistry inside all cells.
If RNA were central to early biochemistry, RNAs must have been able to copy themselves in order for those cells to multiply and evolve. Finding such an RNA copier “is the bull’s-eye of the RNA world hypothesis,” says Gerald Joyce, a chemist at the Scripps Research Institute in San Diego, California. Modern cells instead have a protein-based enzyme called RNA polymerase (RNAP) that copies strands of DNA into their RNA equivalent. In 1993, researchers led by Jack Szostak at Harvard University created an all-RNA version of RNAP, also known as an RNAP ribozyme, which joined two small pieces of RNA on a separate template RNA strand. Since then, Szostak’s team and other have continued to improve their RNA copiers. Two years ago, for example, researchers in the United Kingdom reported isolating an RNAP ribozyme capable of stitching together RNAs up to 200 nucleotides long, again when matching them up to a template strand.
The problem with all of these RNAP ribozymes, Joyce notes, is that they are finicky. They can copy only certain sequences of nucleotide bases, the building blocks that make up RNA and DNA, and those sequences don’t carry out any important function inside cells. So Joyce and his postdoctoral assistant David Horning attempted to come up with a more versatile RNAP ribozyme, using a well-known technique known as in vitro evolution.
They started by synthesizing a large library of DNA strands intended to encode the starting RNAP ribozyme. But they randomly mutated the DNA sequence, ensuring each of the final RNAPs would be different. They added these RNAPs to a vial containing small RNA snippets they wanted to link together on another template RNA strand. If the RNAP ribozyme successfully created a new RNA, the new strand would signal that by binding to a specific molecular target in its vial. And because each RNAP ribozyme was engineered to remain tethered to its new, synthesized RNA strand, this allowed the team to isolate any successes. Each captured RNAP ribozyme was then used as the starting point for another round of evolution.
After 24 rounds of this test tube evolution, in which the scientists successively upped the requirements for what a RNAP ribozyme had to do to be successful, they wound up with one called 24-3 polymerase. That RNA strand, they report online today in the Proceedings of the National Academy of Sciences, is able to copy almost any other RNA, from small catalysts to long RNA based enzymes. The 24-3 polymerase was also able to make copies of RNAs it had already copied, allowing it to amplify the presence of particular RNAs 10,000-fold. That provided the first RNA version of the polymerase chain reaction, a widely used technique to make copies of DNA.
“This paper is an important breakthrough in an ongoing effort to complete the ‘RNA first’ model for the origin of life,” says Steven Benner, an origin-of-life chemist at the Foundation for Applied Molecular Evolution in Alachua, Florida. But Benner cautions that a true confirmation of the RNA world remains a ways off. Not only does 24-3 polymerase’s tightly wound structure prevent it from being able to copy itself, but Benner notes that it has taken the chemistry community 25 years to come up with an RNA copier proficient at copying other RNAs, despite all the tools of modern biochemistry. “[That] suggests we are still missing something important,” Benner says.
Joyce agrees and notes that even if an RNA world preceded the rise of DNA and proteins, it too may have been preceded by earlier forms of biochemistry. Nevertheless, Joyce adds, he and Horning are pressing on to improve 24-3 polymerase further in hopes of making a version that can copy itself. If they succeed, Joyce says, such a molecule could then become the basis for the first synthetic cells that use RNA as the sole genetic information molecule.